Conformational Flexibility Enhances Sensitivity in Fluorescein-Based Voltage Indicators

Voltage imaging with fluorescent indicators offers a powerful complement to traditional electrode or Ca²⁺-imaging approaches to monitoring electrical activity. Small molecule fluorescent indicators present the unique opportunity for exquisite control over molecular structure, enabling detailed investigations of structure/function relationships. In this paper, we examine the use of conformationally-restricted aniline donors within the context of photoinduced electron transfer (PeT) based voltage indicators. We describe the design and synthesis of four new voltage-sensitive fluorophores (VoltageFluors, or VF dyes). We pair these new indicators with existing VF dyes to construct a library of voltage indicators with varying conformations about the dihedral angle between the nitrogen lone pair and the aromatic ring. Using a combination of steady-state and time-resolved fluorescence spectroscopy, cellular electrophysiology, fluorescence lifetime imaging microscopy (FLIM), and functional imaging in mammalian neurons and human cardiomyocytes, we show that differences in voltage sensitivity within aniline-modified VF dyes span an order of magnitude. Annulated anilines show high levels of PeT and low voltage sensitivity, while sterically-congested anilines whose lone pairs do not couple to the aromatic π system are exceptionally bright and display little voltage sensitivity. Conformationally flexible anilines possess the highest absolute voltage sensitivity. Measured using FLIM in patch-clamped HEK cells, we find that voltage sensitivity of the fluorescence lifetime provides the best predictor of performance in cellular systems. By comparing in-depth photophysical characterization with performance in action potential detection, we establish a detailed link between probe conformation and the ability to report on membrane potential dynamics with high fidelity.